Chimeric primers with hairpin conformations and methods of using same

ABSTRACT

Methods and compositions for nucleic acid amplification, detection, and genotyping techniques are disclosed. In one embodiment, a nucleic acid molecule having a target-specific primer sequence; an anti-tag sequence 5′ of the target-specific primer sequence; a tag sequence 5′ of the anti-tag sequence; and a blocker between the anti-tag sequence and the tag sequence is disclosed. Compositions containing such a nucleic acid molecule and methods of using such a nucleic acid molecule are also disclosed.

This application claims priority to U.S. Provisional Application Ser.No. 61/221,271 filed on Jun. 29, 2009, the entirety of which isincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of genetics andmolecular biology. More particularly, it concerns methods andcompositions for the amplification and detection of nucleic acids.

2. Description of Related Art

Nucleic acid amplification and detection techniques are frequentlyemployed in analyzing DNA samples for mutations and polymorphisms. Theyare also employed in the detection and typing of bacteria, virus, andfungi, including those that are infectious pathogens, by analysis oftheir DNA or RNA. Approaches such as allele-specific PCR (AS-PCR) andallele-specific primer extension (ASPE) detect mutations andpolymorphisms using oligonucleotide primers selected such that theyselectively achieve primer extension of either a sequence containing avariant nucleotide or the corresponding sequence containing thewild-type nucleotide. Such approaches are described in, for example,U.S. Pat. Nos. 5,595,890, 5,639,611, and 5,137,806, the disclosures ofwhich are incorporated by reference.

U.S. application Ser. No. 12/262,842, which is incorporated byreference, describes methods and compositions that can simplify geneticanalysis by methods such as the allele-specific primer extension (ASPE)and allele-specific PCR (AS-PCR) methods mentioned above. In certainembodiments, the '842 application employs a primer with a tag sequence5′ of the target specific sequence, and a capture complex comprising ananti-tag sequence complementary to the primer's tag sequence in a methodfor a “one-step” assay. The '842 application discloses that its one-stepamplification and detection methods can reduce the multiple assay stepsin the current commercially available Luminex Tag-It® technologyplatform to a single-step.

Despite the usefulness of the above-mentioned techniques, better methodsof nucleic acid amplification and detection that can provide assays thatrequire less optimization of primer concentrations; provide quickerresults; have lower non-specific background and higher specific signalwhen using DNA binding dyes; provide more sensitive detection ingeneral; provide a more perfect representation of product/targetconcentration; and allow higher multiplexing of primer sets are needed.The methods and composition of the present invention meet these needs asdescribed below.

SUMMARY OF THE INVENTION

The methods and compositions of the present invention provide nucleicacid amplification, detection, and genotyping techniques. In oneembodiment, the present invention provides a nucleic acid moleculecomprising: a target-specific primer sequence; an anti-tag sequence 5′of the target-specific primer sequence; a tag sequence 5′ of theanti-tag sequence; and a blocker between the anti-tag sequence and thetag sequence.

In another embodiment, the present invention provides a compositioncomprising: a microsphere; a first anti-tag nucleic acid covalentlyattached to the microsphere; a tag nucleic acid hybridized to the firstanti-tag nucleic acid; a blocker covalently attached 3′ of the tagsequence; a second anti-tag nucleic acid, which has an identicalsequence to the first anti-tag nucleic acid, covalently attached 3′ ofthe blocker; a target-specific nucleic acid covalently attached 3′ ofthe second anti-tag nucleic acid; and a nucleic acid molecule hybridizedto the second anti-tag nucleic acid and the target-specific nucleicacid, wherein the nucleic acid molecule comprises a sequence that iscomplementary to the sequence of the anti-tag nucleic acid and thetarget-specific nucleic acid.

In another embodiment, the present invention provides a compositioncomprising: (a) a first nucleic acid molecule, wherein the first nucleicacid molecule is a first member of a primer pair, comprising: (i) afirst target-specific primer sequence; (ii) an anti-tag sequence 5′ ofthe target-specific primer sequence; (iii) a tag sequence 5′ of theanti-tag sequence; and (iv) a blocker between the anti-tag sequence andthe tag sequence; and (b) a second nucleic acid molecule, wherein thesecond nucleic acid molecule is a second member of a primer pair,comprising: (i) a second target-specific primer sequence; (ii) auniversal anti-tag sequence 5′ of the target-specific primer sequence;(iii) a universal tag sequence 5′ of the anti-tag sequence; and (iv) ablocker between the anti-tag sequence and the tag sequence; and (c) athird nucleic acid molecule comprising: (i) a universal anti-tagsequence complementary to the universal tag sequence; and (ii) a label.

In other embodiments, the present invention provides a compositioncomprising a plurality of primer pairs for the amplification of aplurality of different target sequences, each primer pair comprising:(a) a first nucleic acid molecule comprising: (i) a firsttarget-specific primer sequence; (ii) an anti-tag sequence 5′ of thetarget-specific primer sequence; (iii) a tag sequence 5′ of the anti-tagsequence; and (iv) a blocker between the anti-tag sequence and the tagsequence; and (b) a second nucleic acid molecule comprising: (i) asecond target-specific primer sequence; (ii) a universal anti-tagsequence 5′ of the target-specific primer sequence; (iii) a universaltag sequence 5′ of the anti-tag sequence; and (iv) a blocker between theanti-tag sequence and the tag sequence; and (c) a labeled, universalanti-tag molecule comprising: (i) a universal anti-tag sequencecomplementary to the universal tag sequence; and (ii) a label.

A composition comprising: (a) a first nucleic acid molecule, wherein thefirst nucleic acid molecule is a first member of a primer pair,comprising: (i) a first target-specific primer sequence; (ii) auniversal anti-tag sequence 5′ of the target-specific primer sequence;(iii) a universal tag sequence 5′ of the anti-tag sequence; and (iv) ablocker between the anti-tag sequence and the tag sequence; (b) a secondnucleic acid molecule, wherein the second nucleic acid molecule is asecond member of a primer pair, comprising: (i) a second target-specificprimer sequence; (ii) a universal anti-tag sequence 5′ of thetarget-specific primer sequence; (iii) a universal tag sequence 5′ ofthe anti-tag sequence; and (iv) a blocker between the anti-tag sequenceand the tag sequence; and (c) a third nucleic acid molecule comprising:(i) a universal anti-tag sequence complementary to the universal tagsequences on the first and second nucleic acid molecules; and (ii) alabel.

In one embodiment, the invention provides a nucleic acid moleculecomprising from 5′ to 3′, a tag region of 24 nucleotide bases, aninternal C18 blocker, a variable length sequence that is complimentaryto a portion of the tag region, and a target-specific primer region,which may be of variable length. The length and composition of thesequence complimentary to a portion of the tag region can be optimizedaccording to buffer composition, hybridization conditions, and thesequence of the tag region. Optimally, the binding of the tag region tothe complimentary sequence should form a hairpin structure withsufficient thermodynamic stability so as to remain in a closed hairpinformation prior to second strand synthesis during amplification, but theenergy barrier should be sufficiently low to allow disruption of thehairpin structure during second strand synthesis.

The tag and anti-tag regions in a hairpin-forming nucleic acid moleculeas described herein may be identical in length or they may be ofdifferent lengths. For example, the tag and anti-tag regions could bothbe 24 nucleotides long, or one region could be 24 nucleotides long whilethe other region is shorter (e.g., 8-16 nucleotides). It can beadvantageous to use tag and anti-tag regions of different lengths inorder to alter the hybridization properties of the hairpin-formingnucleic acid molecule. Preferably, the hairpin region of the molecule isdesigned such that it has a strong enough binding energy to remain inthe closed state until the formation of a double-stranded ampliconproduct causes the hairpin region to open, but a weak enough bindingenergy so as to remain in the open state in the presence of the doublestranded product. Another consideration is the strength of the bindingbetween the tag region of a primer and the anti-tag region used tocapture and/or label (e.g., an anti-tag sequence immobilized on a bead)an amplicon synthesized from the primer. A person of skill in the artwill be familiar with factors affecting DNA hybridization, such assequence length and G+C content, and will be able to determine theappropriate lengths for the tag and anti-tag regions in ahairpin-forming nucleic acid molecule in order to achieve the propertiesmentioned above for a particular application.

In one embodiment, a nucleic acid molecule is provided that comprises,from 5′ to 3′, a tag region of 24 nucleotide bases followed by aninternal C18 blocker, which is then followed by 12 bases that are notcomplimentary to other nucleic acids in the reaction, followed by 12bases that are complimentary to the first 12 bases of the tag region,followed by a target-specific primer region, which may be of variablelength.

In one embodiment, a nucleic acid molecule is provided that comprises,from 5′ to 3′, a tag region of 24 nucleotide bases followed by aninternal C18 blocker, which is then followed by 12 bases that arecomplimentary to the first 12 bases of the tag region, followed by atarget-specific primer region, which may be of variable length.

A target nucleic acid may be any nucleic acid of interest, and thesample containing the target nucleic acid may be any sample thatcontains or is suspected of containing nucleic acids. In certain aspectsof the invention the sample is, for example, from a subject who is beingscreened for the presence or absence of one or more genetic mutations orpolymorphisms. In another aspect of the invention the sample may be froma subject who is being tested for the presence or absence of a pathogen.Where the sample is obtained from a subject, it may be obtained bymethods known to those in the art, such as aspiration, biopsy, swabbing,venipuncture, spinal tap, fecal sample, or urine sample. In certainembodiments the subject is a mammal, bird, or fish. The mammal may be,for example, a human, cat, dog, cow, horse, sheep, swine, swine, rabbit,rat, or mouse. In some aspects of the invention, the sample is anenvironmental sample such as a water, soil, or air sample. In otheraspects of the invention, the sample is from a plant, bacteria, virus,fungi, protozoan, or metazoan.

A primer is a nucleic acid that is capable of priming the synthesis of anascent nucleic acid in a template-dependent process. A target-specificprimer refers to a primer that has been designed to prime the synthesisof a particular target nucleic acid. A primer pair refers to twoprimers, commonly known as a forward primer and a reverse primer or asan upstream primer and a downstream primer, which are designed toamplify a target sequence between the binding sites of the two primerson a template nucleic acid molecule. In certain embodiments, the primerhas a target-specific sequence that is between 10-40, 15-30, or 18-26nucleotides in length.

Various aspects of the present invention use sets of complementary tagand anti-tag sequences. The tags and anti-tags are preferably non-crosshybridizing, i.e., each tag and anti-tag should hybridize only to itscomplementary partner, and not to other tags or anti-tags in the samereaction. Preferably, the tags and anti-tags also will not hybridize toother nucleic acids in the sample during a reaction. The properselection of non-cross hybridizing tag and anti-tag sequences is usefulin assays, particularly assays in a highly parallel hybridizationenvironment, that require stringent non-cross hybridizing behavior. Incertain embodiments, the tag and anti-tag sequences are between 6 to 60,8 to 50, 10 to 40, 10 to 20, 12 to 24, or 20 to 30 nucleotides inlength. In some embodiments, the tag and anti-tag sequences are 12, 14,16, or 24 nucleotides in length. A number of tag and tag complement(i.e., anti-tag) sequences are known in the art and may be used in thepresent invention. For example, U.S. Pat. No. 7,226,737, incorporatedherein by reference, describes a set of 210 non-cross hybridizing tagsand anti-tags. In addition, U.S. Published Application No. 2005/0191625,incorporated herein by reference, discloses a family of 1168 tagsequences with a demonstrated ability to correctly hybridize to theircomplementary sequences with minimal cross hybridization. A “universal”tag or anti-tag refers to a tag or anti-tag that has the same sequenceacross all reactions in a multiplex reaction.

A blocker is a moiety that inhibits extension of the nascent nucleicacid sequence during second strand synthesis. Non-limiting examples ofblocker moieties include C6-20 straight chain alkylenes, iSp18 (which isan 18-atom hexa-ethyleneglycol), iMe-isodC, a hexethylene glycolmonomer, synthetic nucleic acid bases, 2-O-alkyl RNA, or anoligonucleotide sequence in the reverse orientation as compared to thetarget specific sequence.

In certain aspects of the invention, a solid support is used. A varietyof solid supports for the immobilization of biomolecules are known. Forexample, the solid support may be nitrocellulose, nylon membrane, glass,activated quartz, activated glass, polyvinylidene difluoride (PVDF)membrane, polystyrene substrates, polyacrylamide-based substrate, otherpolymers, copolymers, or crosslinked polymers such as poly(vinylchloride), poly(methyl methacrylate), poly(dimethyl siloxane),photopolymers (which contain photoreactive species such as nitrenes,carbenes and ketyl radicals capable of forming covalent links withtarget molecules). A solid support may be in the form of, for example, abead (microsphere), a column, or a chip. Molecules immobilized on planarsolid supports are typically identified by their spatial position on thesupport. Molecules immobilized on non-planar solid supports, such asbeads, are often identified by some form of encoding of the support, asdiscussed below.

Beads may be encoded such that one subpopulation of beads can bedistinguished from another subpopulation. Encoding may be by a varietyof techniques. For example, the beads may be fluorescently labeled withfluorescent dyes having different emission spectra and/or differentsignal intensities. In certain embodiments, the beads are LuminexFlexMAP™ microspheres or Luminex xMAP® microspheres. The size of thebeads in a subpopulation may also be used to distinguish onesubpopulation from another. Another method of modifying a bead is toincorporate a magnetically responsive substance, such as Fe₃O₄, into thestructure. Paramagnetic and superparamagnetic microspheres havenegligible magnetism in the absence of a magnetic field, but applicationof a magnetic field induces alignment of the magnetic domains in themicrospheres, resulting in attraction of the microspheres to the fieldsource. Combining fluorescent dyes, bead size, and/or magneticallyresponsive substances into the beads can further increase the number ofdifferent subpopulations of beads that can be created.

In certain aspects of the invention, the composition comprises aplurality of anti-tag nucleic acid molecules covalently attached to aplurality of encoded microspheres, wherein the plurality of anti-tagmolecules comprise anti-tag sequences that are complementary to the tagsequences in the plurality of primer pairs, and wherein the identity ofeach of the anti-tag nucleic acid molecules can be determined from theencoding of the encoded microsphere to which it is covalently attached.

Nucleic acids in the methods and compositions described herein may belabeled with a reporter. A reporter is a molecule that facilitates thedetection of a molecule to which it is attached. Numerous reportermolecules that may be used to label nucleic acids are known. Directreporter molecules include fluorophores, chromophores, and radiophores.Non-limiting examples of fluorophores include, a red fluorescentsquarine dye such as2,4-Bis[1,3,3-trimethyl-2-indolinylidenemethyl]cyclobutenediylium-1,3-dioxolate,an infrared dye such as2,4Bis[3,3-dimethyl-2-(1H-benz[e]indolinylidenemethyl)]cyclobutenediylium-1,3-dioxolate,or an orange fluorescent squarine dye such as2,4-Bis[3,5-dimethyl-2-pyrrolyl]cyclobutenediylium-1,3-diololate.Additional non-limiting examples of fluorophores include quantum dots,Alexa Fluor® dyes, AMCA, BODIPY® 630/650, BODIPY® 650/665, BODIPY®-FL,BODIPY®-R6G, BODIPY®-TMR, BODIPY®-TRX, Cascade Blue®, CyDye™, includingbut not limited to Cy2™, Cy3™, and Cy5™, a DNA intercalating dye,6-FAM™, Fluorescein, HEX™, 6-JOE, Oregon Green® 488, Oregon Green® 500,Oregon Green® 514, Pacific Blue™, REG, phycobilliproteins including, butnot limited to, phycoerythrin and allophycocyanin, Rhodamine Green™,Rhodamine Red™, ROX™, TAMRA™, TET™, Tetramethylrhodamine, or Texas Red®.A signal amplification reagent, such as tyramide (PerkinElmer), may beused to enhance the fluorescence signal. Indirect reporter moleculesinclude biotin, which must be bound to another molecule such asstreptavidin-phycoerythrin for detection. In a multiplex reaction, thereporter attached to the primer or the dNTP may be the same for allreactions in the multiplex reaction if the identities of theamplification products can be determined based on the specific locationor identity of the solid support to which they hybridize.

In other embodiments, methods for amplifying a target nucleic acid areprovided, which comprise: (a) providing a first primer pair comprising:(i) a first primer comprising: a first target-specific primer sequence;an anti-tag sequence 5′ of the target-specific primer sequence; a tagsequence 5′ of the anti-tag sequence; and a blocker between the anti-tagsequence and the tag sequence; and (ii) a second primer comprising: asecond target-specific primer sequence; (b) providing a reporter; (c)providing a capture complex comprising an anti-tag sequence attached toa solid support; (d) amplifying the target nucleic acid by combining thefirst primer pair, the reporter, the capture complex, and a samplecomprising the target nucleic acid under conditions suitable foramplification of the target nucleic acid. In certain aspects, thereporter is attached to the second primer. In other aspects, thereporter is attached to a dNTP. In yet other embodiments, the reporteris a DNA intercalator. In some embodiments, the method further compriseshybridizing the amplified target nucleic acid to the anti-tag sequenceof the capture complex. In still further embodiments, the method furthercomprises detecting the hybridized, amplified target nucleic acid.Detecting the amplified nucleic acid may comprise, for example, imagingthe amplified target nucleic acid sequence bound to the capture complex.In some embodiments, the sample comprises at least a second targetnucleic acid, and at least a second primer pair is combined with thefirst primer pair, the reporter, the capture complex, and the samplecomprising the target nucleic acids under conditions suitable foramplification of the target nucleic acids. The different amplifiedtarget nucleic acids may be hybridized to different anti-tag sequencesof distinguishable capture complexes. The capture complexes may be, forexample, spatially distinguishable and/or optically distinguishable.

The hairpin forming primers disclosed herein can also be used to amplifyand detect target nucleic acid sequences without the use of a capturecomplex. For example, in one embodiment a target nucleic acid can beamplified by a method comprising: (a) providing a first primer paircomprising: (i) a first target-specific primer sequence; an anti-tagsequence 5′ of the target-specific primer sequence; a tag sequence 5′ ofthe anti-tag sequence; a blocker between the anti-tag sequence and thetag sequence; and a chromophore attached to the tag sequence; and (ii) asecond primer comprising: a second target-specific primer sequence; ananti-tag sequence 5′ of the target-specific primer sequence; a tagsequence 5′ of the anti-tag sequence; a blocker between the anti-tagsequence and the tag sequence; and a chromophore attached to the tagsequence; and (b) a label nucleic acid molecule comprising: (i) ananti-tag sequence complementary to the universal tag sequences of thefirst primer pair; and (ii) a chromophore capable of Forster ResonanceEnergy Transfer with the chromophores of the first primer pair; and (d)amplifying the target nucleic by combining the first primer pair, theuniversal label nucleic acid molecule, and a sample comprising thetarget nucleic acid under conditions suitable for amplification of thetarget nucleic acid. The method may further comprise detecting theamplified nucleic acid. The detection may comprise detecting the FRETbetween the chromophores of the first primer pair and the chromophore ofthe universal label. In certain embodiments, the detection is performedin real-time (i.e., the method provides a real-time PCR). Thisamplification method can be multiplexed, wherein the sample comprises atleast a second target nucleic acid and a second primer pair. Formultiplexed applications, each primer pair and corresponding labelnucleic acid molecule need to have different tag and anti-tag sequencesfrom any other primer pairs and label nucleic acid molecules in thereaction. Additionally, the labels with different emission wavelengthsneed to be used for each different primer pair in the reaction. In someembodiments the primer pair is a nested primer pair and the targetnucleic acid is itself an amplicon.

The hairpin forming probes disclosed herein may also be used to detectthe products of cleavage reactions. Several cleavage-based assays forthe detection of nucleic acid sequences are know in the art. Forexample, Invader technology, which uses a structure-specific flapendonuclease (FEN) to cleave a three-dimensional complex formed byhybridization of allele-specific overlapping oligonucleotides to targetDNA containing a single nucleotide polymorphism (SNP) (single nucleotidepolymorphism) site, is well-known for use in SNP discrimination. Mungbean nuclease and S1 nuclease are also known for their use in SNPdiscrimination because of their ability to cleave single basemismatches. In one embodiment, the present invention provides a methodfor detecting a cleavage product of a nucleic acid cleavage reactioncomprising: (a) providing an oligonucleotide probe comprising: (i) acleavage-product specific sequence; (ii) an anti-tag sequence 5′ of thecleavage-product specific sequence; (iii) a tag sequence 5′ of theanti-tag sequence; (iv) a blocker between the anti-tag sequence and thetag sequence; and (v) a label; (b) hybridizing the oligonucleotide probeto the cleavage product; (c) extending the cleavage product so as todisplace the tag sequence from its hybridization to the anti-tagsequence; (d) hybridizing a labeled anti-tag probe to the displaced tagsequence and (e) detecting the hybridization and extension of theoligonucleotide probe to the cleavage product. The label may be, forexample, a FRET donor or acceptor molecule. This method may be performedwith or without immobilizing the oligonucleotide probe on a solidsupport. In embodiments where the probe is immobilized, theimmobilization may be achieved by hybridization of the tag sequence ofthe probe to a complementary anti-tag sequence coupled to a solidsupport (e.g., a bead or planar array). As discussed above, cleavageproducts may be created by a variety of technologies including, withoutlimitation, those that employ a structure-specific flap endonuclease, amung bean nuclease, or an S1 nuclease. Those of skill in the art will beable to design probes that are susceptible to cleavage when hybridizedto particular target sequence.

Additionally, the hairpin forming probes disclosed herein may be used todetect the formation of a ligation product. A ligation product can beformed when two oligonucleotide probes bind adjacent to one another on atarget nucleic acid. Typically, the ligation is achieved using a ligaseenzyme. In one embodiment, the present invention provides a method fordetecting a ligation product comprising: (a) providing anoligonucleotide probe comprising: (i) a ligation-product specificsequence; (ii) an anti-tag sequence 5′ of the ligation-product specificsequence; (iii) a tag sequence 5′ of the anti-tag sequence; (iv) ablocker between the anti-tag sequence and the tag sequence; and (v) alabel; (b) hybridizing the oligonucleotide probe to the ligation productat a temperature at which the ligation product hybridizes to theolignucleotide probe but at which unligated subunits of the ligationproduct do not hybridize to the oligonucleotide probe; (c) extending theligation product so as to displace the tag sequence from itshybridization to the anti-tag sequence; (d) hybridizing a labeledanti-tag probe to the displaced tag sequence and (e) detecting thehybridization and extension of the oligonucleotide probe to the ligationproduct. The label may be, for example, a FRET donor or acceptormolecule. This method may be performed with or without immobilizing theoligonucleotide probe on a solid support. In embodiments where the probeis immobilized, the immobilization may be achieved by hybridization ofthe tag sequence of the probe to a complementary anti-tag sequencecoupled to a solid support (e.g., a bead or planar array). Those ofskill in the art will be able to design probes that can be ligatedtogether to create a ligation product when hybridized to particulartarget sequence.

In one embodiment, the present invention provides a method forquantifying gene expression comprising: reverse transcribing mRNA from atarget gene to form cDNA; hybridizing to the cDNA a firstoligonucleotide comprising a universal primer-binding sequence and acDNA-specific sequence, and a second oligonucleotide comprising a uniqueprimer-binding sequence and a cDNA-specific sequence; ligating the firstoligonucleotide to the second oligonucleotide to form a ligatedoligonucleotide; amplifying the ligated oligonucleotide using auniversal primer and a unique primer, the unique primer comprising aunique primer sequence, an anti-tag sequence 5′ of the unique primersequence, a tag sequence 5′ of the anti-tag sequence, and a blockerbetween the anti-tag sequence and the tag sequence; capturing theamplicon by hybridizing the tag sequence of the amplicon to an anti-tagsequence of a capture complex; labeling the captured amplicon; anddetecting and quantifying the labeled, captured amplicon. The cDNA canoptionally be immobilized if a wash steps is performed after the reversetranscription reaction. Methods for ligation-mediated amplification areknown in the art and described in, for example, Peck et al., GenomeBiology, 7:R61 (2006), which is incorporated herein by reference. In thecontext of a multiplexed reaction in which the expression of multipletarget genes is analyzed, the “universal primer” and “universalprimer-binding sequence” refer to a common primer and a sequencecomplementary thereto used to analyze all targets in the reaction. Incertain embodiments, the universal primer is a T3 primer. In contrast,the “unique primer” and “unique primer-binding sequence” refer to aprimer and a sequence complementary thereto that is specific for eachdifferent target gene being analyzed. The “unique primer” and “uniqueprimer-binding sequence,” however, are not complementary to a sequencein the target gene itself. The “unique primer” and “uniqueprimer-binding sequence” may be a tag/anti-tag set, but should not becomplementary to any other tag or anti-tag sequence in a multiplexedreaction. In multiplexed reactions, for each of the plurality ofdifferent targets being assayed, there is a unique combination of tagsequence, anti-tag sequence, and capture complex, which will permit theamplicons for each target to be distinguished from that of every othertarget.

The cDNA may be immobilized by methods known to those in the art. Inparticular embodiments, the cDNA is immobilized by capturing and reversetranscribing the mRNA on an oligo-dT coated well or bead.

In other embodiments, the present invention provides a method ofdetecting microorganisms in a sample comprising: (a) providing aplurality of primer pairs for the amplification of a plurality ofdifferent target nucleic acid sequences from a plurality of differentmicroorganisms, each primer pair comprising: (i) a first primercomprising: a first target-specific primer sequence; an anti-tagsequence 5′ of the target-specific primer sequence; a tag sequence 5′ ofthe anti-tag sequence; and a blocker between the anti-tag sequence andthe tag sequence; and (ii) a second primer comprising: a secondtarget-specific primer sequence; a universal anti-tag sequence 5′ of thetarget-specific primer sequence; a universal tag sequence 5′ of theanti-tag sequence; and a blocker between the anti-tag sequence and thetag sequence; (b) providing labeled, universal anti-tag moleculescomprising: (i) a universal anti-tag sequence complementary to theuniversal tag sequence; and (ii) a label; (c) providing a plurality ofcapture complexes comprising anti-tag sequences attached to a solidsupport; (d) amplifying the target nucleic acid sequences from thedifferent microorganisms, if the microorganisms are present in thesample, by combining the plurality of primer pairs, the labeled,universal anti-tag molecule, the capture complexes, and the sample underconditions suitable for amplification of the target nucleic acidsequences; (e) hybridizing the amplified target nucleic acid sequencesto their respective anti-tag sequences of their respective capturecomplexes; and (f) detecting the microorganisms present in the sample bydetecting the amplified target nucleic acid sequences bound to theirrespective capture complexes.

The microorganism may be, for example, a bacteria, virus, retrovirus, orfungus. In certain embodiments, the microorganism is a pathogen. Thesample that contains or may contain the microorganism may be a patientsample, such as a blood sample, serum sample, cerebral spinal fluidsample, stool sample, broncoalveolar lavage sample, sputum, pericardialfluid, peritoneal fluid, pleural fluid, urine, gastric aspirate,abscess, tracheal aspirate, bronchial washing, bone marrow, tissue, etc.In other embodiments, the sample is an environmental sample, such as awater sample or a soil sample. In certain embodiments, between 2 to 100primer pairs are provided for detecting 2 to 100 differentmicroorganisms. Each primer pair may be designed to detect a differentmicroorganism or there can be some redundancy in which two or moreprimer pairs are designed to detect the same microorganism. Typically ina clinical/diagnostic setting or in an environmental setting only asubset of the microorganisms being screened for are expected to bepresent in patient sample or the environmental sample. For example,while a patient sample may be screened for 30 different microorganisms,the patient sample will likely contain only about 0 to 2 of thesemicroorganisms, as it is uncommon for someone to be infected with alarge number of different microorganism at the same time. In certainaspects of the invention, between 0 to 10, 1 to 10, 1 to 5, or 1 to 3different microorganisms are detected.

The amplification may be qualitative, semi-quantitative, orquantitative. In certain embodiments, the amplification may be monitoredin real time (e.g., real-time PCR). When amplification is by thepolymerase chain reaction (PCR), a polymerase possessing stranddisplacement activity should be used as such a polymerase will be ableto open the hairpin structure formed by the hybridization of the tag andanti-tag regions of the primer. In some embodiments, the polymerase isan exo(−) polymerase.

Certain embodiments of the invention comprise the detection of theamplified target nucleic acid. Detection of the amplified target nucleicacid may be by a variety of techniques. In one aspect of the invention,the amplified target nucleic acids are detected using a flow cytometer.Flow cytometry is particularly well-suited where the solid support ofthe capture complex is a bead or other particle. In other aspects of theinvention, detecting the amplified target nucleic acid comprises imagingthe amplified target nucleic acid sequence bound to the capture complex.The imaging may be on, for example, a bead array platform or a chiparray platform.

The methods of the present invention may be used in multiplexed assays.In such multiplexed assay, the sample will typically comprise at least asecond target nucleic acid sequence. In certain aspects of theinvention, there are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240,260, 280, 300, 400, 500, 600, 700, 800, 900, 1000, or any rangederivable therein, target nucleic acid sequences in the sample. Asmentioned above, a target nucleic acid sequence may be any sequence ofinterest. One target nucleic acid sequence may be in the same gene or adifferent gene as another target nucleic acid sequence, and the targetnucleic acid sequences may or may not overlap. Of course, a targetnucleic acid sequence need not be within a gene but may be within, forexample, a non-coding region of DNA. In a multiplex assay where at leasta second target nucleic acid to be amplified is present in a sample, atleast a second discriminating primer or a second primer pair is combinedwith the first primer pair.

An advantage of the methods described herein, is that they may beperformed in a “closed tube” format. In a “closed tube” assay allreagents and sample are added at the start of the reaction, thuseliminating the need for opening of the reaction vessel to add reagentsafter the reaction is initiated. This typically results in a faster turnaround time and reduces the opportunities for contamination and humanerror. Such “closed tube” assays are particularly well-suited forPoint-of-Care applications in which rapid results, minimum humanmanipulations of the assay, and a sterile environment are desirable.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more, unless specifically noted.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A shows an illustration of a hairpin-forming primer and a capturecomplex. FIG. 1B shows an amplification product in which theamplification opened the structure of the hairpin-forming primer suchthat the tag region of the primer is able to hybridize to the anti-tagregion of the capture complex.

FIG. 2 illustrates how when non-hairpin-forming primers are used, thetag regions of non-extended primers can compete with the tag regions ofthe extended primers for hybridization to the anti-tag region of thecapture complex.

FIGS. 3A and 3B show a hairpin-forming forward primer, a hairpin-formingreverse primer, a labeled universal anti-tag molecule, and a capturecomplex before the amplification product is produced (FIG. 3A) and afterthe amplification product is produced (FIG. 3B).

FIGS. 4A to 4E show various configurations of donor and acceptorchromophores in a FRET-based labeling system.

FIGS. 5A to 5B show various configurations of fluorophores and quenchersin a fluorophore/quencher-based labeling system.

FIG. 6 is a graph showing that hairpin primers are more effective in thepresence of excess, non-extended primers than are primers that do notform hairpins.

FIG. 7 is a graph showing a comparison of 12-, 14-, and 16-mer stemhairpin primers, and non-hairpin forming primers (TIF), in a PCR withQiagen HotStart polymerase.

FIG. 8 is a graph showing a comparison of 12-, 14-, and 16-mer stemhairpin primers, and non-hairpin forming primers (TIF), in a PCR withaptaTaq exo(−) polymerase.

FIG. 9 is a graph of the MFI at various PCR cycles in a pseudo real-timePCR with either a hairpin-forming forward primer or a non-hairpinforming forward primer.

FIG. 10 is a graph of the MFI at various PCR cycles in a real-time PCR.

FIG. 11 is a graph representing a dilution series of NeiseriaMeningitidis DNA in a real-time quantitative PCR.

FIG. 12 illustrates a real-time PCR assay chemistry. Upon extension ofthe primers the hairpin portions open up allowing binding to a labeledprobe, such that Forster Resonance Energy Transfer (FRET) occursallowing real-time detection in standard real-time thermal cyclers. Anadvantage of this chemistry is that the end-users need only design theprimers, once they are provided with the validated hairpin sequence,making it very design friendly. No beads are required in this assay.

FIG. 13 illustrates an assay format in which hairpin-forming probes areused with an Invader assay. In the Invader assay the flap portion of theprobe (B) is cleaved. Flap portion (B) can then act as a primer that canopen up the hairpin sequence by polymerase extension. With the tagregion of the hairpin sequence now available for binding it may bind toa labeled probe in a FRET pair for beadless real-time detection or itmay bind to a bead for high multiplex detection.

FIG. 14 illustrates an assay that incorporates the use of a ligationmechanism, such that the assay is held at a high enough temperature sothat probes A and B cannot hybridize to the hairpin primer/probe unlessthey are ligated. Once they are ligated, they are of sufficient bindingstrength to bind to the probe/primer and extend in the presence of astrand displacement polymerase. With the tag region of the hairpinsequence now available for binding it may bind to a labeled probe in aFRET pair for headless real-time detection or it may bind to a bead forhigh multiplex detection.

FIG. 15 illustrates an assay that incorporates the use of a mung bean orS1 nuclease, which has the ability to cleave single base mismatches.Once the mismatch is cleaved, B can now act as a primer that candisplace the hairpin, allowing the tag to be exposed for binding to aprobe, which may or may not be attached to a solid surface.

FIG. 16 illustrates an assay in which universal hairpin primers arecombined with target specific hairpin primers for use as a nestedreal-time PCR assay chemistry. Upon extension of the primers the hairpinportions will open up allowing binding to a labeled probe, such thatFRET occurs allowing real-time detection in standard real-time thermalcyclers. The advantage of this solution is that the end-users need onlydesign the primers, making it very design friendly. No beads arerequired in this embodiment.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. Nucleic Acids

A. Primers

Primers used in the methods and compositions described herein aredesigned to provide better nucleic acid amplification and detection thanpreviously available. Assays that use these primers require lessoptimization of primer concentrations; yield results more quickly;result in lower background and higher specific signal when using DNAbinding dyes; provide greater sensitivity; provide a more accuratemeasure of product/target concentration; and allow higher multiplexingof primer sets. The term “primer,” as used herein, is meant to encompassany nucleic acid that is capable of priming the synthesis of a nascentnucleic acid in a template-dependent process. Primers may be provided indouble-stranded and/or single-stranded form, although thesingle-stranded form is preferred.

In certain embodiments, the methods and compositions disclosed hereinemploy a hairpin-forming primer that, in addition to the target-specificprimer sequence, comprises a tag region and a region that iscomplimentary to the tag region (anti-tag). The tag and anti-tag regionsare separated by a blocker (to prevent polymerase extension into the tagregion). These primers may also be referred to as being “chimeric”because they are composed of regions that serve different purposes.Prior to amplification, the tag and anti-tag regions hybridize forming ahairpin structure, thus sequestering the tag region. Once adouble-stranded amplification product is formed, the hairpin stemstructure is disrupted and the tag region becomes available to bind toanother anti-tag probe, such as an anti-tag probe immobilized on asubstrate (e.g., a bead). An example in which the hairpin-forming primeris the forward primer is illustrated in FIGS. 1A and 1B. It will beunderstood by those in the art that in an alternative embodiment thereverse primer could be the hairpin-forming primer.

As shown in FIG. 1A, the hairpin-forming forward primer comprises atarget-specific primer region, an anti-tag region, a blocker region (aC18 spacer in this drawing), and a tag region. The anti-tag region ofthe primer can be the same length as the tag region or it can be adifferent length. In FIG. 1A the anti-tag region is shorter than itscomplementary tag region; thus it is referred to as a partial anti-tagregion. Prior to polymerase extension and the creation of adouble-stranded amplification product, the anti-tag region hybridizeswith the tag region to form a hairpin structure, which prevents the tagregion on the primer from hybridizing to the anti-tag region that iscoupled to the bead. As shown in FIG. 1B, upon extension of the reverseprimer, a polymerase with strand displacement activity will disrupt thehairpin stem and stop at the blocker allowing the tag region tohybridize to the anti-tag region on the bead.

Unextended forward primers will be inhibited from binding theimmobilized anti-tag probes because of sequestration of the tag regionsin a hairpin structure. This is advantageous because the occupation ofhybridization sites on capture complexes by unextended primers can limitthe availability of capture probes for labeled amplification product andthus decrease assay sensitivity. This is particularly problematic earlyin an amplification reaction due to the high ratio of unextended primersto extended primers at this stage. This effect is most significant whentrying to measure accumulation of amplified product in real-time. Asillustrated in FIG. 2A, excess unextended tagged primers that do notform hairpins can compete with the amplification products forhybridization sites on the capture complexes. Moreover, if intercalatingor DNA binding dyes are used, they will bind to the double-strandednucleic acid created by the hybridization of the unextended primer tothe probe causing an increase in background signal. In contrast, whenusing primers with a hairpin structure, the primers and probes will nothybridize until a PCR amplification product is formed.

The use of primers as described above can provide at least the followingbenefits, as compared to the use of non-hairpin forming primers: (1)requires less optimization of primer concentration; (2) produces fasterresults because fewer PCR cycles are required to achieve detectablesignal; (3) produces lower background and higher specific signal whenusing DNA binding dyes; (4) provides more sensitive detection ingeneral; and (5) provides a more accurate representation ofproduct/target concentration.

In certain embodiments, both the forward and the reverse primer of aprimer pair are hairpin-forming primers. This can be particularlyadvantageous in multiplexed reactions. In this case, one of the primersof the primer pair comprises universal tag and anti-tag sequences. Thetag and anti-tag sequences are “universal” because, while thetarget-specific primer sequence varies for each different target in themultiplexed amplification reaction, the same (i.e. “universal”) tag andanti-tag sequences are used. An example illustrated in FIGS. 3A and 3Bshow a hairpin-forming forward primer comprising a target-specificprimer region, and complementary anti-tag and tag regions separated by ablocker region. Also, shown is a hairpin-forming reverse primercomprising a target-specific primer region, and universal anti-tag andtag regions separated by a blocker region. The forward primer andreverse primer are designed such that they will prime the synthesis of adouble-stranded nucleic acid during the polymerase chain reaction. In amultiplexed reaction, the anti-tag region and tag region of the forwardprimer are unique for each different forward primer in the reaction. Inthis way, amplification products of the extended forward primer can beidentified by hybridization to a probe sequence. The universal anti-tagregion and universal tag region, however, are the same for all reverseprimers in the reaction. This allows the labeled, universal anti-tagprobe to label all extended, reverse primers in the reaction. Thisgreatly reduces the amount of label (e.g., fluorophore) required. Forexample, in a 30-plex PCR panel for infectious diseases in which 30different reverse primers are directly labeled, 6,000 nM of fluorophorewould be required, whereas only 200 nM of fluorophore would be requiredwith a labeled, universal anti-tag. This is a 30× reduction in theamount of reporter required. These calculations are based on a 30-plexpanel in which a sample is expected to test positive for 0 to 2infectious agents. The ability to use less label reduces the backgroundof the assay, reduces the amount of reagents needed, and can eliminatethe need for a wash step to remove excess label from the assay.

B. Preparation of Nucleic Acids

The nucleic acids disclosed herein may be prepared by any techniqueknown to one of ordinary skill in the art, such as for example, chemicalsynthesis, enzymatic production, or biological production. Non-limitingexamples of a synthetic nucleic acid (e.g., a syntheticoligonucleotide), include a nucleic acid made by in vitro chemicalsynthesis using phosphotriester, phosphite or phosphoramidite chemistryand solid phase techniques such as described in EP 266,032, incorporatedherein by reference, or via deoxynucleoside H-phosphonate intermediatesas described by Froehler et al., 1986 and U.S. Pat. No. 5,705,629, eachincorporated herein by reference. Various different mechanisms ofoligonucleotide synthesis have been disclosed in for example, U.S. Pat.Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, 5,428,148,5,554,744, 5,574,146, 5,602,244, each of which is incorporated herein byreference.

A non-limiting example of an enzymatically produced nucleic acid includeone produced by enzymes in amplification reactions such as PCR™ (see forexample, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, eachincorporated herein by reference), or the synthesis of anoligonucleotide described in U.S. Pat. No. 5,645,897, incorporatedherein by reference. A non-limiting example of a biologically producednucleic acid includes a recombinant nucleic acid produced (i.e.,replicated) in a living cell, such as a recombinant DNA vectorreplicated in bacteria (see for example, Sambrook et al., 2001,incorporated herein by reference).

Nucleic acids used as a template for amplification may be isolated fromcells, tissues or other samples according to standard methodologies(Sambrook et al., 2001). In certain embodiments, analysis is performedon whole cell or tissue homogenates or biological fluid samples withoutsubstantial purification of the template nucleic acid. The nucleic acidmay be genomic DNA or fractionated or whole cell RNA. Where RNA is used,it may be desired to first convert the RNA to a complementary DNA(cDNA).

Depending upon the desired application, high stringency hybridizationconditions may be selected that will only allow hybridization betweensequences that are completely complementary. In other embodiments,hybridization may occur under reduced stringency to allow foramplification of nucleic acids containing one or more mismatches withthe primer sequences. Once hybridized, the template-primer complex iscontacted with one or more enzymes that facilitate template-dependentnucleic acid synthesis. Multiple rounds of amplification, also referredto as “cycles,” are conducted until a sufficient amount of amplificationproduct is produced.

A reverse transcriptase PCR™ amplification procedure may be performed toreverse transcribe mRNA into cDNA. Methods of RT-PCR are well known inthe art (see Sambrook et al., 2001). Alternative methods for RT-PCRutilize thermostable DNA polymerases. These methods are described in WO90/07641. Polymerase chain reaction methodologies are well known in theart. Representative methods of RT-PCR are described in U.S. Pat. No.5,882,864.

Another method for amplification is ligase chain reaction (“LCR”),disclosed in European Application No. 320 308, incorporated herein byreference in its entirety. U.S. Pat. No. 4,883,750 describes a methodsimilar to LCR for binding probe pairs to a target sequence. A methodbased on PCR™ and oligonucleotide ligase assay (OLA), disclosed in U.S.Pat. No. 5,912,148, may also be used.

Alternative methods for amplification of nucleic acid sequences that maybe used in the practice of certain aspects of the present invention aredisclosed in U.S. Pat. Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546,5,849,497, 5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574,5,928,905, 5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, GBApplication No. 2 202 328, and in PCT Application No. PCT/US89/01025,each of which is incorporated herein by reference in its entirety.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, mayalso be used as an amplification method in the present invention. Inthis method, a replicative sequence of RNA that has a regioncomplementary to that of a target is added to a sample in the presenceof an RNA polymerase. The polymerase will copy the replicative sequence,which may then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention (Walker et al., 1992). StrandDisplacement Amplification (SDA), disclosed in U.S. Pat. No. 5,916,779,is another method of carrying out isothermal amplification of nucleicacids, which involves multiple rounds of strand displacement andsynthesis, i.e., nick translation.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989; Gingeras et al., PCTApplication WO 88/10315, incorporated herein by reference in theirentirety). European Application No. 329 822 disclose a nucleic acidamplification process involving cyclically synthesizing single-strandedRNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA).

PCT Application WO 89/06700 (incorporated herein by reference in itsentirety) disclose a nucleic acid sequence amplification scheme based onthe hybridization of a promoter region/primer sequence to a targetsingle-stranded DNA (“ssDNA”) followed by transcription of many RNAcopies of the sequence. This scheme is not cyclic, i.e., new templatesare not produced from the resultant RNA transcripts. Other amplificationmethods include “race” and “one-sided PCR™” (Frohman, 1990; Ohara etal., 1989).

Amplification products may be visualized. If the amplification productsare integrally labeled with radio- or fluorescent-labeled nucleotides,the amplification products can be exposed to x-ray film or visualizedunder the appropriate excitatory spectra. In another approach, a labelednucleic acid probe is hybridized to the amplification product. The probemay be conjugated to, for example, a chromophore, fluorophore,radiolabel, or conjugated to a binding partner, such as an antibody orbiotin.

Various nucleic acid detection methods known in the art are disclosed inU.S. Pat. Nos. 5,840,873, 5,843,640, 5,843,651, 5,846,708, 5,846,717,5,846,726, 5,846,729, 5,849,487, 5,853,990, 5,853,992, 5,853,993,5,856,092, 5,861,244, 5,863,732, 5,863,753, 5,866,331, 5,905,024,5,910,407, 5,912,124, 5,912,145, 5,919,630, 5,925,517, 5,928,862,5,928,869, 5,929,227, 5,932,413 and 5,935,791, each of which isincorporated herein by reference.

C. Hybridization

Sequence-specific nucleic acid hybridization assays are used for thedetection of specific genetic sequences as indicators of geneticanomalies, mutations, and disease propensity. In addition, they are usedfor the detection of various biological agents and infectious pathogens.As used herein, “hybridization,” “hybridizes” or “capable ofhybridizing” is understood to mean the forming of a double or triplestranded molecule or a molecule with partial double or triple strandednature. The term “anneal” as used herein is synonymous with “hybridize.”The term “hybridization,” “hybridizes” or “capable of hybridizing”encompasses the terms “stringent conditions” or “high stringency” andthe terms “low stringency” or “low stringency conditions.”

As used herein “stringent conditions” or “high stringency” are thoseconditions that allow hybridization between or within one or morenucleic acid strands containing complementary sequences, but precludehybridization of non-complementary sequences. Such conditions are wellknown to those of ordinary skill in the art, and are preferred forapplications requiring high selectivity. Stringent conditions maycomprise low salt and/or high temperature conditions, such as providedby about 0.02 M to about 0.15 M NaCl at temperatures of about 50° C. toabout 70° C. It is understood that the temperature and ionic strength ofa desired stringency are determined in part by the length of theparticular nucleic acids, the length and nucleobase content of thetarget sequences, the charge composition of the nucleic acids, and tothe presence or concentration of formamide, tetramethylammonium chlorideor other solvents in a hybridization mixture.

It is also understood that these ranges, compositions and conditions forhybridization are mentioned by way of non-limiting examples only, andthat the desired stringency for a particular hybridization reaction isoften determined empirically by comparison to one or more positive ornegative controls. Non-limiting examples of low stringency conditionsinclude hybridization performed at about 0.15 M to about 0.9 M NaCl at atemperature range of about 20° C. to about 50° C. Of course, it iswithin the skill of one in the art to further modify the low or highstringency conditions to suit a particular application.

II. Detection of Nucleic Acids

A. Labels

To detect nucleic acids, it will be advantageous to employ nucleic acidsin combination with an appropriate detection system. Recognitionmoieties incorporated into primers, incorporated into the amplifiedproduct during amplification, or attached to probes are useful in theidentification of nucleic acid molecules. A number of different labels,also referred to as “reporters,” may be used for this purpose such asfluorophores, chromophores, radiophores, enzymatic tags, antibodies,chemi/electroluminescent labels, affinity labels, etc. One of skill inthe art will recognize that these and other labels not mentioned hereincan be used with success in this invention. Examples of affinity labelsinclude, but are not limited to the following: an antibody, an antibodyfragment, a receptor protein, a hormone, biotin, digoxigen, DNP, or anypolypeptide/protein molecule that binds to an affinity label.

Examples of enzyme tags include enzymes such as urease, alkalinephosphatase or peroxidase to mention a few. Colorimetric indicatorsubstrates can be employed to provide a detection means visible to thehuman eye or spectrophotometrically, to identify specific hybridizationwith complementary nucleic acid-containing samples. All of theseexamples are generally known in the art and the skilled artisan willrecognize that the invention is not limited to the examples describedabove.

Examples of fluorophores include, a red fluorescent squarine dye such as2,4-Bis[1,3,3-trimethyl-2-indolinylidenemethyl]cyclobutenediylium-1,3-dioxolate,an infrared dye such as2,4Bis[3,3-dimethyl-2-(1H-benz[e]indolinylidenemethyl)]cyclobutenediylium-1,3-dioxolate,or an orange fluorescent squarine dye such as2,4-Bis[3,5-dimethyl-2-pyrrolyl]cyclobutenediylium-1,3-diololate.Additional non-limiting examples of fluorophores include quantum dots,Alexa Fluor® dyes, AMCA, BODIPY® 630/650, BODIPY® 650/665, BODIPY®-FL,BODIPY®-R6G, BODIPY®-TMR, BODIPY®-TRX, Cascade Blue®, CyDye™, includingbut not limited to Cy2™, Cy3™, and Cy5™, a DNA intercalating dye,6-FAM™, Fluorescein, HEX™, 6-JOE, Oregon Green® 488, Oregon Green® 500,Oregon Green® 514, Pacific Blue™, REG, phycobilliproteins including, butnot limited to, phycoerythrin and allophycocyanin, Rhodamine Green™,Rhodamine Red™, ROX™, TAMRA™, TET™, Tetramethylrhodamine, or Texas Red®.A signal amplification reagent, such as tyramide (PerkinElmer), may beused to enhance the fluorescence signal.

It is contemplated that FRET-based detection systems may be used withthe methods and compositions disclosed herein. FRET (fluorescenceresonance energy transfer or Forster resonance energy transfer) makesuse of the transfer of energy between donor and acceptor chromophores.In certain embodiments, a chromophore is attached to the hairpinsequence or the blocker and another chromophore is attached to thecapture complex, such that upon attachment of the primer to the capturecomplex, an increase in signal will be observed by virtue of the energytransfer between the donor and acceptor chromophores. Various,non-limiting examples of the configurations of the donor and acceptorchromophores are shown in FIGS. 4A to 4E. FRET-based detection reducebackground and therefore allow for higher multiplexing of primer setscompared to free floating chromophore methods, particularly in closedtube and real-time detection systems.

It is also contemplated that fluorophore/quencher-based detectionsystems may be used with the methods and compositions disclosed herein.When a quencher and fluorophore are in proximity to each other, thequencher quenches the signal produced by the fluorophore. Aconformational change in the nucleic acid molecule separates thefluorophore and quencher to allow the fluorophore to emit a fluorescentsignal. Various, non-limiting examples of the configurations of thefluorophore and quencher are shown in FIGS. 5A to 5B. Like FRET-baseddetection, fluorophore/quencher-based detection systems reducebackground and therefore allow for higher multiplexing of primer setscompared to free floating fluorophore methods, particularly in closedtube and real-time detection systems.

B. Gene Chips and Microarrays

Certain embodiments of the present invention involve a solid support.The solid support may be a planar array, such as a gene chip ormicroarray. Arrays and gene chip technology provide a means of rapidlyscreening a large number of nucleic acid samples for their ability tohybridize to a variety of single stranded oligonucleotide probesimmobilized on a solid substrate. These techniques involve quantitativemethods for analyzing large numbers of genes rapidly and accurately. Thetechnology capitalizes on the complementary binding properties of singlestranded DNA to screen DNA samples by hybridization (Pease et al., 1994;Fodor et al., 1991). Basically, an array or gene chip consists of asolid substrate upon which an array of single stranded DNA or RNAmolecules have been attached. For screening, the chip or array iscontacted with a single stranded DNA or RNA sample, which is allowed tohybridize under stringent conditions. The chip or array is then scannedto determine which probes have hybridized. The identity of the probes onthe chip or planar array is known by its spatial location (i.e., x, ycoordinate) on the chip or planar array.

The ability to directly synthesize on or attach polynucleotide probes tosolid substrates is well known in the art. See U.S. Pat. Nos. 5,837,832and 5,837,860, both of which are expressly incorporated by reference. Avariety of methods have been utilized to either permanently or removablyattach the probes to the substrate. Exemplary methods include: theimmobilization of biotinylated nucleic acid molecules toavidin/streptavidin coated supports (Holmstrom, 1993), the directcovalent attachment of short, 5′-phosphorylated primers to chemicallymodified polystyrene plates (Rasmussen et al., 1991), or the precoatingof the polystyrene or glass solid phases with poly-L-Lys or poly L-Lys,Phe, followed by the covalent attachment of either amino- orsulfhydryl-modified oligonucleotides using bi-functional crosslinkingreagents (Running et al., 1990; Newton et al., 1993). When immobilizedonto a substrate, the probes are stabilized and therefore may be usedrepeatedly. In general terms, hybridization is performed on animmobilized nucleic acid target or a probe molecule that is attached toa solid surface such as nitrocellulose, nylon membrane or glass.Numerous other matrix materials may be used, including reinforcednitrocellulose membrane, activated quartz, activated glass,polyvinylidene difluoride (PVDF) membrane, polystyrene substrates,polyacrylamide-based substrate, other polymers such as poly(vinylchloride), poly(methyl methacrylate), poly(dimethyl siloxane),photopolymers (which contain photoreactive species such as nitrenes,carbenes and ketyl radicals capable of forming covalent links withtarget molecules.

C. Bead Arrays

In some embodiments, the solid support may be a microsphere.Microsphere-based assays may also be analyzed by technologies known tothose in the art. For example, in certain embodiments, Luminex xMAP®technology may be used. The Luminex technology allows the detection ofnucleic acid products immobilized on fluorescently encoded microspheres.By dyeing microspheres with 10 different intensities of each of twospectrally distinct fluorochromes, 100 fluorescently distinctpopulations of microspheres are produced. These individual populations(sets) can represent individual detection sequences and the magnitude ofhybridization on each set can be detected individually. The magnitude ofthe hybridization reaction is measured using a third reporter, which istypically a third spectrally distinct fluorophore. The reporter moleculesignals the extent of the reaction by attaching to the molecules on themicrospheres. As both the microspheres and the reporter molecules arelabeled, digital signal processing allows the translation of signalsinto real-time, quantitative data for each reaction. The Luminextechnology is described, for example, in U.S. Pat. Nos. 5,736,330,5,981,180, and 6,057,107, all of which are specifically incorporated byreference.

Flow cytometry can be used for simultaneous sequence identification andhybridization quantification in microsphere-based assays. Internal dyesin the microspheres are detected by flow cytometry and used to identifythe specific nucleic acid sequence to which a microsphere is coupled.The label on the target nucleic acid molecule is also detected by flowcytometry and used to quantify target hybridization to the microsphere.Methods of flow cytometry are well know in the art and are described,for example, in U.S. patents, all of which are specifically incorporatedby reference. U.S. Pat. Nos. 5,981,180, 4,284,412; 4,989,977; 4,498,766;5,478,722; 4,857,451; 4,774,189; 4,767,206; 4,714,682; 5,160,974; and4,661,913

Microspheres may also be analyzed on array platforms that image beadsand analytes distributed on a substantially planar array. In this way,imaging of bead arrays is similar to the gene chips discussed above.However, in contrast to gene chips where the analyte is identified byits spatial position on the array, bead arrays typically identify theanalyte by the encoded microsphere to which it is bound. Examples ofcommercially available bead array systems include Illumina's BeadXpress™Reader and BeadStation 500™.

D. Competitive Binding Assays

Embodiments of the present invention may also be used in conjunctionwith a competitive binding assay format. In general, this formatinvolves a sequence coupled to a solid surface, and a labeled sequence,which is complementary to the sequence coupled to the solid surface, insolution. With this format, the target sequence in the sample beingassayed does not need to be labeled. Rather, the target sequence'spresence in the sample is detected because it competes with the labeledcomplement for hybridization with the immobilized detection sequence.Thus, if the target sequence is present in the sample, the signaldecreases as compared to a sample lacking the target sequence. TheLuminex xMAP technology described above can be used in a competitivebinding assay format. The use of the Luminex technology in a competitivebinding assay format is described in U.S. Pat. Nos. 5,736,330 and6,057,107, incorporated herein by reference.

E. Tag Sequences

As mentioned above, various aspects of the present invention usecomplementary tag sequences (i.e., tags and anti-tags). A number ofapproaches have been developed that involve the use of oligonucleotidetags attached to a solid support that can be used to specificallyhybridize to their tag complements that are coupled to primers, probesequences, target sequences, etc. The proper selection of non-crosshybridizing tag and anti-tag sequences is useful in assays, particularlyassays in a highly parallel hybridization environment, that requirestringent non-cross hybridizing behavior.

Certain thermodynamic properties of forming nucleic acid hybrids areconsidered in the design of tag and anti-tag sequences. The temperatureat which oligonucleotides form duplexes with their complementarysequences known as the T_(m) (the temperature at which 50% of thenucleic acid duplex is dissociated) varies according to a number ofsequence dependent properties including the hydrogen bonding energies ofthe canonical pairs A-T and G-C (reflected in GC or base composition),stacking free energy and, to a lesser extent, nearest neighborinteractions. These energies vary widely among oligonucleotides that aretypically used in hybridization assays. For example, hybridization oftwo probe sequences composed of 24 nucleotides, one with a 40% GCcontent and the other with a 60% GC content, with its complementarytarget under standard conditions theoretically may have a 10° C.difference in melting temperature (Mueller et al., 1993). Problems inhybridization occur when the hybrids are allowed to form underhybridization conditions that include a single hybridization temperaturethat is not optimal for correct hybridization of all oligonucleotidesequences of a set. Mismatch hybridization of non-complementary probescan occur forming duplexes with measurable mismatch stability(Santalucia et al., 1999). Mismatching of duplexes in a particular setof oligonucleotides can occur under hybridization conditions where themismatch results in a decrease in duplex stability that results in ahigher T_(m) than the least stable correct duplex of that particularset. For example, if hybridization is carried out under conditions thatfavor the AT-rich perfect match duplex sequence, the possibility existsfor hybridizing a GC-rich duplex sequence that contains a mismatchedbase having a melting temperature that is still above the correctlyformed AT-rich duplex. Therefore, design of families of oligonucleotidesequences that can be used in multiplexed hybridization reactions mustinclude consideration for the thermodynamic properties ofoligonucleotides and duplex formation that will reduce or eliminatecross hybridization behavior within the designed oligonucleotide set.

There are a number of different approaches for selecting tag andanti-tag sequences for use in multiplexed hybridization assays. Theselection of sequences that can be used as zip codes or tags in anaddressable array has been described in the patent literature in anapproach taken by Brenner and co-workers (U.S. Pat. No. 5,654,413,incorporated herein by reference). Chetverin et al. (WO 93/17126, U.S.Pat. Nos. 6,103,463 and 6,322,971, incorporated herein by reference)discloses sectioned, binary oligonucleotide arrays to sort and surveynucleic acids. These arrays have a constant nucleotide sequence attachedto an adjacent variable nucleotide sequence, both bound to a solidsupport by a covalent linking moiety. Parameters used in the design oftags based on subunits are discussed in Barany et al. (WO 9731256,incorporated herein by reference). A multiplex sequencing method hasbeen described in U.S. Pat. No. 4,942,124, incorporated herein byreference. This method uses at least two vectors that differ from eachother at a tag sequence.

U.S. Pat. No. 7,226,737, incorporated herein by reference, describes aset of 210 non-cross hybridizing tags and anti-tags. U.S. PublishedApplication No. 2005/0191625, incorporated herein by reference,discloses a family of 1168 tag sequences with a demonstrated ability tocorrectly hybridize to their complementary sequences with minimal crosshybridization.

A population of oligonucleotide tag or anti-tag sequences may beconjugated to a population of primers or other polynucleotide sequencesin several different ways including, but not limited to, direct chemicalsynthesis, chemical coupling, ligation, amplification, and the like.Sequence tags that have been synthesized with primer sequences can beused for enzymatic extension of the primer on the target for example inPCR amplification. A population of oligonucleotide tag or anti-tagsequences may be conjugated to a solid support by, for example, surfacechemistries on the surface of the support.

8. Blocker Moieties

Blocker moieties prevent the polymerase from extending through the tagsequence region during second strand synthesis, thus allowing the tagsequence to remain single-stranded during amplification and thereforefree to hybridize to its complementary anti-tag sequence in the capturecomplex.

A blocker moiety refers to any moiety that when linked (e.g., covalentlylinked) between a first nucleotide sequence and a second nucleotidesequence is effective to inhibit and preferably prevent extension ofeither the first or second nucleotide sequence but not both the firstand second nucleotide sequence. There are a number of molecules that maybe used as blocker moieties. Non-limiting examples of blocker moietiesinclude C6-20 straight chain alkylenes and iSp18 (which is an 18-atomhexa-ethyleneglycol). Blocker moieties may include, for example, atleast one deoxy ribofuranosyl naphthalene or ribofuranosyl naphthalenemoiety, which may be linked to the adjacent nucleotides via a3′-furanosyl linkage or preferably via a 2′-furanosyl linkage. A blockermoiety may be an oligonucleotide sequence that is in the oppositeorientation as the target specific sequence. Various blocker moietiesand their use are described in U.S. Pat. No. 5,525,494, which isincorporated herein by reference.

III. Examples

The following examples are included to demonstrate certain embodimentsof the invention. Those of skill in the art should, in light of thepresent disclosure, will appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

A. Example 1

A side by side study was performed to compare hairpin-forming primerscomprising tag and anti-tag regions with non-hairpin forming primerscomprising a tag region but not an anti-tag region. These “tagged”primers were used as forward primers. Cy3-labeled reverse primers (400nM) were added along with the respective forward primer (hairpin formingor non-hairpin forming) as well as a detection bead prior to PCRamplification. The target was a thrombophilia gene, MTHFR Exon 7. After27 cycles, the samples were analyzed on an LX200 (Luminex Corp.) at roomtemperature without any other addition of buffer or reporter, thusrepresenting a simulated closed-tube detection format.

The forward primers were:

12snap: (SEQ ID NO: 1) 5′CAAACAAACATTCAAATATCAATC/iSp18/CTATCTATACATAATGTTTGTTTGCAAGGAGGAGCTGCTGAAGATG3′ 12isoSNAP: (SEQ ID NO: 2)5′CAAACAAACATTCAAATATCAATC/ie-isodC//iMeisodC/CTATCTATACATAATGTTTGTTTGCAAGGAGGAGCTGCTGAAGATG3′. Snap 1: (SEQ ID NO: 3)5′CAAACAAACATTCAAATATCAATC/iSp18/GATTGATATTGAATGTTGTTTGCAAGGAGGAGCTGCTCAACATG3′ No Snap (SEQ ID NO: 4)5′CAAACAAACATTCAAATATCAATC/iSp18/CTATCTATACATTTACAAACATTCCAAGGAGGAGCTGCTGAAGATG3′ TIF (SEQ ID NO: 5)5′CAAACAAACATTCAAATATCAATC/iSp18/CAAGGAGGAGCTGCT CAACATG3′The sequence of the Cy3-labeled reverse primer was:

/5Cy3/CACTTTGTGACCATTCCGGTTTG (SEQ ID NO: 6)

12snap and 12 isoSNAP differed from each other in that 12snap containsan iSp18 blocker and 12 isoSNAP contains an isodC blocker. The 12snapand 12 isoSNAP were calculated to be in the hairpin conformation 99.9%of the time in solution (37° C.; [monovalent]=0.0500 mol/L;[Mg²⁺]=0.0015 mol/L; [Betaine]=1.00 mol/L) by Visual OMP software. Snap1was similar to 12snap except that it had 24-base tag hybridizing to itscomplimentary anti-tag in the hairpin, rather than 12 bases as in12snap. NoSnap was as long as 12snap but without a hairpin structure.The TIF primer included a tag sequence but no complementary anti-tagsequence.

All PCR reactions were performed in the same cocktail. These wereanalyzed after 27 cycles of amplification and 36 cycles ofamplification. A PCR cocktail was prepared using the followingconcentrations and reagents:

TABLE 1 lx Volume Master Mix 25 μl HotStart TAQ Plus Master Mix 2X(Qiagen) H₂O 21 μl RNASE FREE Water (Qiagen) Primer  1 μl IDT (400 nMfinal concentration each) Template  1 μl Purified Human DNA Sample fromUCLA (100 ng) Beads  2 μl MagPlex Microspheres (Luminex) (5000 per set)Total 50 μl

These formulations were used along with the downstream primer:

(SEQ ID NO: 7) LUA-MED-TF/5Cy3/CACTTTGTGACCATTCCGGTTTG.

Each primer was at 400 nM concentration. The cycling conditions for thisreaction were as follows:

Heat Denaturation Step; 95° C. for 5 min.

Cycling Steps (for 36 cycles): 94° C. for 30 s, 55° C. for 30 s, 72° C.for 30 s.

After amplification the reactions were stored until all reactions werecompleted and were then placed in a v-bottom plate and analyzed on aLuminex 200 analyzer after 3 minutes at 96° C. and 12 minutes at 37° C.

Results are shown in Table 2 below. Analyte 27 is the positive bead setwith the anti-tag region coupled to the bead. Analyte 33 is the negativecontrol with a non-specific sequence coupled to the bead. Median valuesare shown.

TABLE 2 Analyte 27 Analyte 33 Location Sample MFI MFI Cycles  1(1, A1)12isoSNAP 77 11 36  2(1, B1) 12isoSNAP 86 4  3(1, C1) 12snap 113 14 4(1, D1) 12snap 120 9.5  5(1, E1) NoSnap 107 13  6(1, F1) NoSnap 111.59.5  7(1, G1) snap1 98.5 10  8(1, H1) snap1 98.5 8  9(1, A2) TIF 118 1210(1, B2) TIF 108 15 11(1, C2) TIF 36 10.5 27 12(1, D2) TIF 32.5 7 13(1,E2) snap1 24.5 7 14(1, F2) snap1 21 13.5 15(1, G2) NoSnap 20 8 16(1, H2)NoSnap 26.5 12.5 17(1, A3) 12snap 58 12 18(1, B3) 12snap 53 15.5 19(1,C3) 12isoSNAP 25 9 20(1, D3) 12isoSNAP 31.5 13

The high signal (MFIs of 58 and 53) from the 12snap primer at the27^(th) cycle as compared to the signal (MFIs of 20 and 26.5) from theNoSnap primer indicates that the 12snap primer is folding when it issupposed to and not interfering with hybridization at the stage of thereaction where excess primer would be expected.

B. Example 2

Another study was performed in which after amplification to 27 cycles asdescribed in Example 1, the samples were spiked with more of the sameforward primer (400 nM) that they were originally amplified with.Addition of excess primer prior to hybridization, but afteramplification, was done to test whether the non-extended hairpin primerswere interfering with hybridization to the bead, and whether the TIFprimers were interfering with hybridization. If there was interference,one would expect that the spiked primers would decrease the MFI value.

TABLE 3 Analyte 27 Analyte 33 Location Sample MFI MFI Total Events 1(1,A1) TIF 91.5 8 203 2(1, B1) TIF 94 5.5 201 3(1, C1) 12snap 115.5 11 2114(1, D1) 12snap 119 8 206 5(1, E1) TIF spiked 400 nM 63 13.5 208 6(1,F1) TIF spiked 400 nM 64 8.5 208 7(1, G1) 12snap spiked 400 nM 106 13207 8(1, H1) 12snap spiked 400 nM 109 15 203

As shown in Table 3 and FIG. 6, the MFI for the TIF primer sampledropped to 68% of its original value when excess TIF primer was added,whereas the 12snap primer only dropped to 92% of its original value whenexcess 12snap primer was added. The slight drop in signal observed withthe 12snap primer could be due to incomplete hairpin structure formationby these primers, as only 12 base pairs are available for hairpinformation.

C. Example 3

Varying amounts of primer concentration were tested in PCR reactionsstopped at 27 cycles to determine whether the signal would increase ordecrease with additional amounts of TIF primer or 12snap primer. Theresults are shown in Tables 4 and 5 below.

TABLE 4 12snap primer Primer Analyte 27 Analyte 33 Total LocationConcentration MFI MFI Events 1(1, A1) 100 nM 96 4 213 2(1, B1) 100 nM 994 203 3(1, C1) 100 nM 101.5 7 211 4(1, D1) 200 nM 111.5 8 220 5(1, E1)200 nM 103 7 212 6(1, F1) 200 nM 108 10 200 7(1, G1) 400 nM 121 12 2068(1, H1) 400 nM 114 8 205 9(1, A2) 400 nM 118.5 14.5 214

TABLE 5 TIF primer Primer Analyte 27 Analyte 33 Location ConcentrationMFI MFI Total Events 1(1, A1) 100 nM 53 2 234 2(1, B1) 100 nM 63.5 5 2143(1, C1) 100 nM 62 3.5 206 4(1, D1) 200 nM 60 5.5 209 5(1, E1) 200 nM 6311 207 6(1, F1) 200 nM 62 7.5 216 7(1, G1) 400 nM 55 13 209 8(1, H1) 400nM 43 8.5 209 9(1, A2) 400 nM 52 14 203

The data show that one can gain greater signal with the hairpin primerby increasing the concentration, whereas greater signal cannot beobtained by increasing the TIF primer. This indicates that the hairpinprimer does not interfere as much with hybridization to the bead as doesthe TIF primer.

D. Example 4

Two additional studies were performed to confirm: (1) that theCy3-labeled reverse primer was not hybridizing to the forward primers;and (2) that primer dimers were not forming.

The primers (the Cy3-labeled reverse primer and either 12snap or TIF)were mixed with the anti-tagged beads in PCR solution and heated to 96°C. for 3 minutes followed by 37° C. for 12 minutes. As shown in Table 6,no non-specific binding of the reverse primer to either forward primerwas detected.

TABLE 6 Analyte Location Sample 27 MFI Analyte 33 MFI Total Events 1(1,A1) 12snap 14.5 24.5 204 2(1, B1) 12snap 12 14 204 3(1, C1) no forwardprimer 3 3.5 203 4(1, D1) TIF 12 10.5 205 5(1, E1) TIF 11 6 215 6(1, F1)no forward primer 2 2.5 200

PCR reactions with the upstream primers 12snap and TIF and theCy3-labeled reverse primer were performed in the absence of template tocheck for the formation of primer dimers. The reactions were run induplicate on RD18 after a 3 minute 96° C. and 12 minute 37° C. hybprotocol. The results shown in Table 7 indicate that no primer dimerswere formed and hybridized to the beads.

TABLE 7 Analyte Total Location Sample 27 MFI Analyte 33 MFI Events  1(1,A1) TIF, no template 0.5 2 205  1(1, B1) TIF, no template 0.5 0 209 6(1, F1) 12snap, no template 5 0.5 203  5(1, E1) 12snap, no template 20 212  9(1, A2) TIF 102.5 0 200 10(1, B2) TIF 104 0 207 14(1, F2) 12snap132 3.5 208 13(1, E2) 12snap 120 0 224

E. Example 5

Forward primers that formed 16-mer and 14-mer stem structures in thehairpins were also studied. An additional tagged, but non-hairpinfowling primer, TIF, was also included in these studies.

16snap: (SEQ ID NO: 8) CAA ACA AAC ATT CAA ATA TCA ATC /iSp18/CTC TCTATT TTG AAT GTT TGT TTG CAA GGA GGA GCT GCT GAA GAT G 14snap:(SEQ ID NO: 9) CAA ACA AAC ATT CAA ATA TCA ATC /iSp18/CTC AACTAT TTT GAA TGT TTG TTT GCA AGG AGG AGC TGC TGA AGA TG

16snap and 14snap were tested in PCR reactions and in PCR solution usingoligos complimentary to the primer region. The following PCR set up wasdesigned to test the oligos in Qiagen Hotstart Master Mix with no extraMgCl₂ added.

TABLE 8 Master Mix 25 μl H₂O 19.75 μl   Primer  2 μl Template 0.25 μl  Beads (2)  3 μl Total 50 μl

The reaction was stopped at 27 cycles and hybridized for 2 minutes at96° C. followed by 37° C. for 12 minutes. The results were as follows:

TABLE 9 Location Sample Analyte 27 MFI Analyte 33 MFI  1(1, A1) 16snap33 0  2(1, B1) 16snap 34 1  3(1, C1) 16snap 35 2  4(1, E1) 14snap 32 2 5(1, F1) 14snap 31 0  6(1, G1) 14snap 34.5 3.5  7(1, A2) 12snap 52.5 3 8(1, B2) 12snap 59 0  9(1, C2) 12snap 62.5 1 10(1, E2) TIF 47.5 0 11(1,F2) TIF 47 0 12(1, G2) TIF 48 2

The data from Table 9 is also represented graphically in FIG. 7. The16snap and 14snap primers produced lower signals than 12snap. They alsoproduced lower signals than TIF. It was postulated that the exonucleaseactivity of the polymerase in the Qiagen Hotstart Master Mix wasdegrading the stem structure on the 16snap and 14snap primers.Accordingly, another PCR, which was also stopped at 27 cycles, wasperformed using the exo (−) polymerase apta taq. The cocktail for thisPCR was as follows:

TABLE 10 10x Buffer 5 μl H₂O 29.5 μl   Primer 4 μl Template 0.25 μl  Beads (2) 2 μl dNTPs 1 μl apta taq 0.25 μl   MgCl₂ 8 μl Total 50 μl 

The results of the reaction with apta taq were as follows:

TABLE 11 Location Sample Analyte 27 MFI Analyte 33 MFI  1(1, A1) 16snap98 0  2(1, B1) 16snap 99 2  3(1, C1) 16snap 94 4  4(1, D1) 16snap 106 2 5(1, E1) 14snap 133 0  6(1, F1) 14snap 127.5 0  7(1, G1) 14snap 130.5 2 8(1, H1) 14snap 125.5 0.5  9(1, A2) 12snap 170 0 10(1, B2) 12snap 157.52.5 11(1, C2) 12snap 162.5 1 12(1, D2) 12snap 162 0.5 13(1, E2) TIF 34.55 14(1, F2) TIF 32 0 15(1, G2) TIF 36 0 16(1, H2) TIF 32 3

The data from Table 11 is also represented graphically in FIG. 8. All ofthe hairpin-forming primers (16snap, 14snap, and 12snap) significantlyoutperformed the non-hairpin-forming primer (TIF).

F. Example 6

The PCR cocktail as described above in the apta taq PCR was used as ahybridization buffer for hybridizing a labeled oligo complimentary tothe primer region for each of the 16snap, 14snap, 12snap, and TIFprimers. As shown in Table 12, the hairpin structure of the 16snap,14snap, and 12snap primers largely inhibited their ability to hybridizeto the beads.

TABLE 12 Location Sample Analyte 27 MFI Analyte 33 MFI 1(1, A1) 16snap12.5 9 2(1, B1) 16snap 10.5 8 3(1, C1) 14snap 16 12 4(1, D1) 14snap 19 95(1, E1) 12snap 35 10.5 6(1, F1) 12snap 35 9.5 7(1, A2) TIF 425 12 8(1,B2) TIF 427 13.5

G. Example 7

Oligos were also made for the amplification of the prothrombin gene.These oligos were as follows:

PT TIF: (SEQ ID NO: 10) CAA TTC AAA TCA CAA TAA TCA ATC /iSp18/CTT CCTGAG CCC AGA GAG C 12SNAP/ptu: (SEQ ID NO: 11)CAA TTC AAA TCA CAA TAA TCA ATC /iSp18/ACA CTCCAC ACATGA TTT GAA TTG CTT CCT GAG CCC AGA GAG C PtdCY3: (SEQ ID NO: 12)/5Cy3/ GTC ATT GAT CAG TTT GGA GAG TAG G BeadPT: (SEQ ID NO: 13)/5AmMC12/GAT TGA TTA TTG TGA TTT GAA TTG FVmutant oligo FV506Q2:(SEQ ID NO: 14) /5AmMC12/GTATTCCTTGCCTGTCCA

The BeadPT oligo was coupled to bead set 29. The Analyte 33 and Analyte27 bead sets from the MTHFR studies described above, were used asnegative controls.

Two PCR cocktails were prepared sharing all the same reagents with theexception of the forward primers (PT TIF and 12SNAP/ptu). A no templatecontrol was added as well as 3 template added samples for eachcondition. The PCR cocktail is shown in Table 13. Results are shown inTable 14.

TABLE 13 10x ThermoPol Buffer 5 μl H₂O 34.7 μl Primer 2 μl (400 nM)Template #24 0.3 μl (100 ng) Beads 2 μl (5,000 beads) dNTPs 1 μl (0.2mM) Deep Vent Polymerase 2 μl (10 Units) (New England BioLabs) MgSO₄ 3μl (8 mM) Total 50 μl

TABLE 14 Analyte Analyte Analyte Total Location Sample 27 MFI 29 MFI 33Events 1(1,A1) 12SNAP/ptu, 14.5 10 23 184 no template 2(1,B1) 12SNAP/ptu10.5 78 12.5 415 3(1,C1) 12SNAP/ptu 6.5 84 14 322 4(1,D1) PT TIF, 7 1617 346 5(1,E1) PT TIF 15 38 14 336 6(1,F1) PT TIF 6 36.5 10.5 193

From the results in Table 14, it can be seen that the signal differenceof the 12SNAP/ptu primer is about double that of the PT TIF primer after27 cycles using 400 nM primer concentrations with a PCR protocol of: 97°C., 5 minutes; (97° C., 30 seconds; 55° C., 30 seconds; 72° C., 30seconds)×27 cycles; followed by 7 minutes at 72° C. These results alsodemonstrated that the PT primers will not cross hybridize with the MHFTRprimer sets if combined into a multiplex reaction.

H. Example 8

A pseudo real-time PCR was performed in which a PCR cocktail (withDeepVent exo (−) polymerase) was divided into 16 aliquots for eachprimer set. Each aliquot was removed from the thermal cycler atprogressive cycles to measure the signal levels at each cycle. This wasdone using a fast 2-step PCR reaction, and aliquots were measured on anLX200 (Luminex) at room temperature at the end of the PCR protocol of 36cycles total.

As shown in FIG. 9 signal was observable at just 22 cycles (44 minutes)for the 12SNAP/ptu primer compared to 29 cycles (58 min.) for the PT TIFprimer. In addition to producing an observable signal earlier, it can beseen in FIG. 9 that the hairpin forming primer also was more sensitive.At cycle 22 the PT TIF primer was only 16% of the signal compared to the12SNAP/ptu primer, and only 75% at peak cycle 33.

I. Example 9

A real-time PCR experiment was performed on a glass slide with the lensand hex-illuminator directly over the slide for the duration of theexperiment. Glass slide chambers were constructed, and the glass waschemically modified using DMDCS followed by a dip in Polyadenylic AcidPotassium salt. A glass slide and a cover slip were joined together witha sticky gasket (BioRad) using an in situ PCR kit. These were placedonto a BioRad DNA Engine thermal cycler equipped with a slide griddle.This particular slide griddle had a hole drilled in it, directly overone of the 96 wells. The exposed well was painted black. The glasschamber was placed directly over the hole in the griddle to reducebackground reflection light. A real-time PCR unit was constructed bycoupling a CCD camera and light source to the DNA Engine thermal cycler.The Hex-illuminator was placed directly over the glass slide andremained there for the duration of the PCR reaction. The followingcocktail was placed in the 25 μL volume glass chamber:

TABLE 15 10x ThermoPol Buffer 5 μl H₂O 34.7 μl Primer 200 nM each12SNAP/ptu & PtdCY3 Template #24 0.3 μl (100 ng) Beads 2 μl (5,000beads) dNTPs 1 μl (0.2 mM each) Deep Vent Polymerase 2 μl (10 Units)(New England BioLabs) MgSO₄ 3 μl (8 mM) Total 50 μl

The following PCR Cycling conditions were run: 1) 97° C. for 5 min; 2)105° C. for 15 s; 3) 96° C. for 30 s; 4) 50° C. for 5 s; 5) 68° C. for30 s; 6) Go to 2, 5 times; 7) 15° C. for 10 s; 8) 24° C. for 5 min; 9)Go to 2, 6 times; 10) End. These conditions included extra ramp times toaccount for the heating delay of the griddle.

Images of the beads were taken at exactly 4 minutes at each 5 cycleinterval at 24° C. The glass chamber was not agitated in between runs.The data are shown in Table 16 and FIG. 10.

TABLE 16 Cycle before Bead MFI 6 1 251 2 232 12 1 246 2 248 18 1 235 2241 24 1 276 2 220 30 1 316 2 183 36 1 281 2 154 42 1 299 2 178

J. Example 10

Optimization of the length of the stem region of the hairpin used in theprimer that contains the universal Tag sequence that binds to theuniversal labeled probe upon extension of the opposite strand wasevaluated. In order to find the optimal length of the reverse primerhairpin stem region length, and the length of the universal reporterprobes, a series of primers with different stem lengths and universalreporter probes with different lengths were reacted for comparison.

In these reactions reverse hairpin primers with 11mer, 14mer, 16mer and0 mer stem lengths were used. These were hybridized with beads that werecoupled to probes that were complimentary to the target specific primerregions of each of these hairpin primers. Each 50 uL reaction contained:

8 mM MgCl₂

1× Qiagen PCR buffer

5000 beads

200 nM of primer

200 nM of universal reporter probe

All reagents were hybridized at 95° C. for 5 min. followed by 37° C. for15 minutes. The Luminex magnetic beads were then analyzed on a LuminexLx200 analyzer.

The following oligos were used in this reaction:

BeadTagantiprime (SEQ ID NO: 15) /5AmMC12/TAG TTG CAA ATC CGC GAC AANoSnaprevNei (SEQ ID NO: 16)ATG ATG ATG TAT TGT AGT TAT GAA /iSp18/AGG TATTGA AGT TTT GTC GCG GAT TTG CAA CTA Univlabeled13 (SEQ ID NO: 17)/5Cy3/AAT ACA TCA TCA T/3InvdT/ UnivLabeled 15 (SEQ ID NO: 18)/5Cy3/ACA ATA CAT CAT CAT /3InvdT/ Snap11revNei (SEQ ID NO: 19)ATG ATG ATG TAT TGT AGT TAT GAA /iSp18/TAC ATCATC ATT TGT CGC GGA TTT GCA ACT A Snap14RevNei (SEQ ID NO: 20)ATG ATG ATG TAT TGT AGT TAT GAA /iSp18/CAA TACATC ATC ATT TGT CGC GGA TTT GCA ACT A Snap16RevNei (SEQ ID NO: 21)ATG ATG ATG TAT TGT AGT TAT GAA /iSpl8/TAC AATACA TCA TCA TTT GTC GCG GAT TTG CAA CTA

TABLE 17 Rev primer type universal probe MFI Snap11revNei Univlabeled138 Snap11revNei Univlabeled13 6 Snap11revNei UnivLabeled 15 40Snap11revNei UnivLabeled 15 43 Snap14RevNei Univlabeled13 3 Snap14RevNeiUnivlabeled13 3 Snap14RevNei UnivLabeled 15 3 Snap14RevNei UnivLabeled15 4 Snap16RevNei Univlabeled13 3 Snap16RevNei Univlabeled13 1Snap16RevNei UnivLabeled 15 2 Snap16RevNei UnivLabeled 15 2 NoSnaprevNeiUnivlabeled13 929 NoSnaprevNei Univlabeled13 1009 NoSnaprevNeiUnivLabeled 15 862 NoSnaprevNei UnivLabeled 15 1399

The goal of this study was to find a primer/probe pair such that thehairpin region of the primer would remain in the closed state in thepresence of the universal labeled probe, but remain in the open stateonce a double stranded amplicon product was formed in the presence ofthe universal labeled probe. In order to ensure that the hairpin wouldremain in the open state after formation of the double stranded product,we chose a primer/probe pair such that the hairpin monomer was of astrong enough binding energy so as to remain in the closed state, but ofa weak enough binding energy so as to remain in the open state in thepresence of the double stranded product and universal labeled probe.Such a pair would have to near the point of open state in this study.The best pair was identified as the Snap11revNei/Univlabeled13 pair.This pair was chosen to be used in subsequent PCR reactions because thelow MFI indicates that it is in the closed state, but if a longeruniversal labeled probe is used, some of the hairpins open, as indicatedby the 40-43 MFIs. This indicates that the Snap11revNei/Univlabeled13 isclosed, but it is near the point at which some would be open.

K. Example 11

A dilution series of Neiseria Meningitidis DNA (ATCC 700532D-5) in areal-time quantitative PCR was performed in a closed tube. Thisexperiment demonstrated the ability to perform quantitative real-timePCR in order to discriminate between varying input concentrations oftemplate DNA. A PCR cocktail was prepared such that each 25 uL reactioncontained:

8 mM MgCl₂

1× Qiagen PCR buffer

5000 beads of each region

200 nM of primer

200 nM of universal reporter probe

A sample of Neiseria Meningitidis DNA was amplified in real-time usingsealed glass chambers and placed on a thermal cycler fitted with a slidegriddle. These reactions were performed as in Example 11. The firstsealed chamber contained 1 million copies of DNA, the second chambercontained 100,000 copies, and the third chamber contained 10,000 copiesof N. Meningitidis DNA.

Two bead sets were used in this experiment. One bead set (Set 2) wascoupled to a probe (BeadTag Nei) that was complimentary to the tagregion of the forward primer (Snap12fwdShrtNei). The other bead set(Set 1) was coupled to a probe (BeadTag antiList) that was not specificto hybridize to anything in the reaction. Set 1 was used to monitor thenon-specific signal in the reaction and to act as a normalization andbackground subtract tool to account for differences in light intensityfor each image taken.

Snap11revNei (SEQ ID NO: 22)ATG ATG ATG TAT TGT AGT TAT GAA /iSp18/TAC ATCATC ATT TGT CGC GGA TTT GCA ACT A BeadTag Nei (SEQ ID NO: 23)/5AmMC12/GAT TGA TAT TTG AAT GTT TGT TTG /3InvdT/ Snap12fwdShrtNei(SEQ ID NO: 24) CAA ACA AAC ATT CAA ATA TCA ATC /iSp18/AAT GTTTGT TTG GCT GCG GTA GGT GGT TCA A Univlabeled13 (SEQ ID NO: 25)AAT ACA TCA TCA T/3Cy3/ BeadTag antiList (SEQ ID NO: 26)/5AmMC12/GTT TGT ATT TAG ATG AAT AGA AAG /3InvdT/

The data for each bead set for each cycle is given below. The averageMFI for each bead Set 1 for each time point in all 3 reactions was 3931MFI. This average value was used to create a normalization factor bydividing the non-specific bead set (Set 1) by 3931 MFI. Each raw datapoint was divided by this normalization factor that was specific to eachtime point. After normalization, the specific bead set for detectingNeiseria Meningitidis (Set 2) normalized MFI was subtracted by Set 1normalized MFI. This calculation resulted in a net normalized MFI forSet 2 for each time point. The data are given in the table below.

TABLE 18 Raw Normalization Net Normalized Net Cycle Bead Set MedianFactor MFI MFI MFI Normalized 1 million copies 0 1 3757 0.96 127 3931133 2 3884 4064 12 1 3120 0.79 −76 3931 −96 2 3044 3836 18 1 3124 0.79158 3931 199 2 3282 4130 24 1 3009 0.77 563 3931 736 2 3572 4667 30 12863 0.73 601 3931 825 2 3464 4757 100,000 copies 0 1 3931 1.00 −15 3931−15 2 3916 3916 6 1 4120 1.05 18 3931 17 2 4138 3949 12 1 4403 1.12 583931 52 2 4461 3983 18 1 4431 1.13 154 3931 137 2 4585 4068 24 1 43961.12 579 3931 518 2 4975 4449 30 1 4083 1.04 790 3931 761 2 4873 4692 361 4175 1.06 787 3931 741 2 4962 4673 10,000 copies 0 1 4220 1.07 1363931 127 2 4356 4058 6 1 4417 1.12 244 3931 217 2 4661 4149 12 1 48191.23 210 3931 171 2 5029 4103 18 1 4671 1.19 144 3931 121 2 4815 4053 241 4671 1.19 144 3931 121 2 4815 4053 30 1 3326 0.85 521 3931 616 2 38474547 36 1 3162 0.80 699 3931 869 2 3861 4801

A graph of this data (FIG. 11) shows a clear distinction between each ofthe input concentrations of N. Meningitidis which allows forquantitation.

L. Example 12

The following results demonstrate the ability to multiplexhairpin-forming primers for the detection of pathogens. A 3-plexmeningitis assay was designed to detect Neisseria Meningitidis, ListeriaMonocytogenes, and Haemophilus Influenzae. Three primer sets weremultiplexed in the same reaction. Genomic DNA from separate bacteriaspecies were placed in individual reactions to demonstrate thespecificity of the assay.

The following primer and probe sequences were ordered from IDT and used.

Primer Set 1: SIF4fwdList-t88 (SEQ ID NO: 27)TTA CTT CAC TTT CTA TTT ACA ATC /iSp18/AAG TGAAGT AAA TTG CGA AAT TTG GTA CAG C SIF13RCrevList (SEQ ID NO: 28)ATG ATG ATG TAT TGT AGT TAT GAA /iSp18/TAC ATCATC ATC TGA TTG CGC CGA AGT TTA CAT TC Primer Set 2: SIFprobeFwdHaem-t86(SEQ ID NO: 29) CTA ATT ACT AAC ATC ACT AAC AAT /iSp18/GTT AGT AAT TAG TTG TTT ATA ACA ACG AAG GGA CTA ACG T  SIFrevHaem(SEQ ID NO: 30) ATG ATG ATG TAT TGT AGT TAT GAA /iSp18/TAC ATC ATC ATG ATT GCG TAA TGC ACC GTG TT  Primer Set 3: Snap12fwdShrtNei(SEQ ID NO: 31) CAA ACA AAC ATT CAA ATA TCA ATC /iSp18/AAT GTTTGT TTG GCT GCG GTA GGT GGT TCA A Snap11revNei (SEQ ID NO: 32)ATG ATG ATG TAT TGT AGT TAT GAA /iSp18/TAC ATCATC ATT TGT CGC GGA TTT GCA ACT A Probes coupled to Beads:Bead Set 27/Specific for N. meningitidis fwd. primer: (SEQ ID NO: 33)/5AmMC12/GAT TGA TAT TTG AAT GTT TGT TTG /3InvdT/Bead Set 62/Specific for L. Monocytogenes fwd. primer: (SEQ ID NO: 34)GAT TGT AAA TAG AAA GTG AAG TAA /3AmM/Bead Set 67/ Specific for H. Influenzae fwd. primer: (SEQ ID NO: 35)ATT GTT AGT GAT GTT AGT AAT TAG /3AmM/ Universal Labeled Probe:(SEQ ID NO: 36) 13Uni - AAT ACA TCA TCA T/3Cy3Sp/

The following volumes in μL were used in each PCR cocktail:

TABLE 19 1x Material volume 10x Buffer 5 H20 35.1 Primer set 1 (10 μM) 1Primer set 2 (10 μM) 1 Primer set 3 (10 μM) 1 Each Template 1 dntps (10mM) 1 Polymerase (50 U/μL) 0.2 MgCl2 (50 mM) 6 Bead Set 1 (5000beads/μL) 0.5 Bead Set 2 (5000 beads/μL) 0.5 Bead Set 3 (5000 beads/μL)0.5 U13 (100 μM) 0.2 TOTAL 53

PCR Materials: (Roche) Apta Taq delta exo DNA pol., Glycerol free, 50U/ul-Sample 2, 5 KU (100 ul); (Roche PN:13409500) PCR Buffer withoutMgCl2, 10× concentration; (Invitrogen PN:18427-088) 10 mM dNTP Mix.

Thermal Cycling Parameters: 97° C. for 4 min; then 35 cycles of: (97° C.for 30 sec, 62° C. for 30 sec); then 72° C. for 7 min.

Each bead set was previously prepared using Luminex MagPlex-C MagneticMicrospheres by coupling their respective probe sequences using Luminexrecommended EDC coupling procedures.

The following genomic DNA samples were obtained from American TypeCulture Collection (ATCC):

TABLE 20 Item Number Description Lot Number 700532D-5 DR Neisseriameningitidis; Strain FAM18 7385221 BAA-679D-5 DR Listeria monocytogenes;Strain EGDe 57878064 51907D FZ Haemophilus influenzae 2662083

After the PCR reaction, samples were heated to 95° C. for 2 minutes andplaced at room temperature for 8 minutes prior to analyzing on a Luminex200 analyzer. 100 bead events per bead set were collected and a MedianFluorescence Intensity (MFI) value was derived for each bead set in eachreaction. The following MFI values were obtained for each sample:

TABLE 21 Input Genomic Bead Set DNA 27 Bead Set 62 Bead Set 67 Sample 1H. Influenza 7 10 67 Sample 2 L. Monocytogenes 6 87 3 Sample 3 N.Meningitidis 73 13.5 0 Sample 4 No Template 2 9.5 0

These results demonstrate the multiplex ability of hairpin primers.

The portions of the primer sets that are target specific to thedifferent bacterial species were obtained from the following publiclyavailable references:

Neisseria Meningitidis:

Corless, C. E., Guiver, M., Borrow, R., Edwards-Jones, V., Fox, A. J.,and Kaczmarski, E. 2001. Simultaneous Detection of NeisseriaMeningitidis, Haemophilus Influenzae, and Streptococcus Pneumoniae inSuspected Cases of Meningitis and Septicemia Using Real-Time PCR.Journal of Clinical Microbiology. 39: 1553-1558.

Listeria monocytogenes:

Johnson, w., Tyler, S., Ewan, E., Ashton, F., Wang, G. and Rozee, K.1992. Detection of Genes Coding for Listeriolysin and Listeriamonocytogenes Antigen A (LmaA) in Listeria spp. by the Polymerase ChainReaction. Microbial Pathogenesis 12; 79-86.

Bohnert, M., Dilasser, F., Dalet, C. Mengaud, J. and Cossart, P. 1992.Use of Specific Oligonucleotides for Direct Enumeration of Listeriamonocytogenes in Food Samples by Colony Hybridization and RapidDetection by PCR. Res. Microbiol. 143; 271-280.

Haemophilus Influenzae:

Maaroufi, Y., Bruyne, J., Heymans, C., and Crokaert, F. 2007. Real-TimePCR for Determining Capsular Serotypes of Haemophilus Influenzae.Journal of Clinical Microbiology. 45: 2305-2308.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe methods described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A nucleic acid molecule comprising: (a) a target-specific primersequence; (b) an anti-tag sequence 5′ of the target-specific primersequence; (c) a tag sequence 5′ of the anti-tag sequence; and (d) ablocker between the anti-tag sequence and the tag sequence.
 2. Thenucleic acid molecule of claim 1, wherein the anti-tag sequence and thetag sequence are each from 8 to 30 nucleotides in length.
 3. The nucleicacid molecule of claim 2, wherein the anti-tag sequence is 12nucleotides in length.
 4. The nucleic acid molecule of claim 2, whereinthe anti-tag sequence and the tag sequence are each 24 nucleotides inlength.
 5. The nucleic acid molecule of claim 1, wherein the blocker isan iSp18 moiety or an iMe-isodC moiety.
 6. (canceled)
 10. A compositioncomprising: (a) a first nucleic acid molecule, wherein the first nucleicacid molecule is a first member of a primer pair, comprising: (i) afirst target-specific primer sequence; (ii) an anti-tag sequence 5′ ofthe target-specific primer sequence; (iii) a tag sequence 5′ of theanti-tag sequence; and (iv) a blocker between the anti-tag sequence andthe tag sequence; (b) a second nucleic acid molecule, wherein the secondnucleic acid molecule is a second member of a primer pair, comprising:(i) a second target-specific primer sequence; (ii) a universal anti-tagsequence 5′ of the target-specific primer sequence; (iii) a universaltag sequence 5′ of the anti-tag sequence; and (iv) a blocker between theanti-tag sequence and the tag sequence; and (c) a third nucleic acidmolecule comprising: (i) a universal anti-tag sequence complementary tothe universal tag sequence; and (ii) a label.
 11. The composition ofclaim 10, wherein the label is a fluorescent label. 12-13. (canceled)14. The composition of claim 10, comprising a plurality of primer pairsfor the amplification of a plurality of different target sequences and aplurality of anti-tag nucleic acid molecules covalently attached to aplurality of encoded microspheres, wherein the plurality of anti-tagmolecules comprise anti-tag sequences that are complementary to the tagsequences in the plurality of primer pairs, and wherein the identity ofeach of the anti-tag nucleic acid molecules can be determined from theencoding of the encoded microsphere to which it is covalently attached.15. A method for amplifying a target nucleic acid comprising: (a)providing a first primer pair comprising: (i) a first primer comprising:a first target-specific primer sequence; an anti-tag sequence 5′ of thetarget-specific primer sequence; a tag sequence 5′ of the anti-tagsequence; and a blocker between the anti-tag sequence and the tagsequence; and (ii) a second primer comprising: a second target-specificprimer sequence; and (b) providing a reporter; (c) providing a capturecomplex comprising an anti-tag sequence attached to a solid support; and(d) amplifying the target nucleic by combining the first primer pair,the reporter, the capture complex, and a sample comprising the targetnucleic acid under conditions suitable for amplification of the targetnucleic acid.
 16. The method of claim 15, further comprising hybridizingthe amplified nucleic acid to the anti-tag sequence of the capturecomplex. 17-22. (canceled)
 23. The method of claim 16, furthercomprising detecting the amplified nucleic acid.
 24. The method of claim23, wherein detecting the amplified nucleic acid comprises imaging theamplified target nucleic acid sequence bound to the capture complex. 25.The method of claim 15, wherein the sample comprises at least a secondtarget nucleic acid and at least a second primer pair is combined withthe first primer pair, the reporter, the capture complex, and the samplecomprising the target nucleic acids under conditions suitable foramplification of the target nucleic acids.
 26. (canceled)
 27. The methodof claim 26, further comprising hybridizing the different amplifiedtarget nucleic acids to different anti-tag sequences of distinguishablecapture complexes.
 28. The method of claim 27, wherein the capturecomplexes are spatially distinguishable or optically distinguishable.29-40. (canceled)
 41. A method for quantifying gene expressioncomprising: (a) immobilizing cDNA reverse transcribed from mRNA; (b)hybridizing to the immobilized cDNA a first oligonucleotide comprising auniversal primer-binding sequence and a cDNA-specific sequence, and asecond oligonucleotide comprising a unique primer-binding sequence and acDNA-specific sequence; (c) ligating the first oligonucleotide to thesecond oligonucleotide to form a ligated oligonucleotide; (d) amplifyingthe ligated oligonucleotide using a universal primer and a uniqueprimer, the unique primer comprising a unique primer sequence, ananti-tag sequence 5′ of the unique primer sequence, a tag sequence 5′ ofthe anti-tag sequence; and a blocker between the anti-tag sequence andthe tag sequence; (e) capturing the amplicon by hybridizing the tagsequence of the amplicon to an anti-tag sequence of a capture complex;(f) labeling the amplicon; and (g) detecting and quantifying thelabeled, captured amplicon.
 42. The method of claim 41, comprisingcapturing and reverse transcribing the mRNA on an oligo-dT coated wellor bead.
 43. The method of claim 41, wherein the universal primer is aT3 primer.
 44. The method of claim 41, wherein the labeling of thecaptured amplicon is with a DNA-binding dye.
 45. The method of claim 41,wherein the expression is quantified for a plurality of different genes,and for each of the different genes there is a different tag, anti-tag,and capture complex.
 46. The method of claim 41, wherein the amplicon islabeled prior to capturing.
 47. The method of claim 41, wherein theamplicon is labeled after capturing. 48-66. (canceled)
 67. A method fordetecting a target nucleic acid comprising: (a) providing anoligonucleotide probe comprising: (i) a target-specific sequence; (ii)an anti-tag sequence 5′ of the target-specific sequence; (iii) a tagsequence 5′ of the anti-tag sequence; (iv) a blocker between theanti-tag sequence and the tag sequence; and (v) a label; (b) hybridizingthe oligonucleotide probe to the target nucleic acid sequence; and (c)detecting the hybridization of the oligonucleotide probe to the targetnucleic acid sequence.
 68. The method of claim 67, wherein the targetnucleic acid sequence is a cleavage product of a nucleic acid cleavagereaction.
 69. The method of claim 68, wherein the cleavage product ismade by a structure-specific flap endonuclease or a mung bean nucleaseor an S1 nuclease.
 70. The method of claim 67, wherein the targetnucleic acid sequence is a ligation product.
 71. The method of claim 67,wherein the label is a FRET donor or acceptor molecule.
 72. The methodof claim 67, further comprising immobilizing the oligonucleotide probeon a solid support.
 73. The method of claim 72, wherein theoligonucleotide probe is immobilized on a solid support by hybridizationof the tag sequence to a complementary anti-tag sequence coupled to thesolid support.